# US 7,410,790 B2 · Method for producing γ-glutamylcysteine

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- Number: US 7,410,790 B2
- Document ID: US7410790B2
- Status: Granted
- Kind: B2
- Application number: 10/732,523
- Filed: 2003-12-11
- Published: 2004-12-23
- Granted: 2008-08-12
- Earliest claimed: 2002-12-13
- Assignees: Ajinomoto Co., Inc.
- Inventors: Mariko Suehiro; Hiroaki Nishiuchi; Yasushi Nishimura
- Web page: https://patentgrep.com/patent/US7410790B2

## CPC

- A23L 2/56: Flavouring or bittering agents (sweeteners A23L2/60)
- A23L 27/24: prepared by fermentation
- A23L 31/00: Edible extracts or preparations of fungi; Preparation or treatment thereof
- A23L 33/145: Extracts
- C12N 9/88: Lyases (4.)
- C12P 13/12: Methionine; Cysteine; Cystine
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The yeast which has γ-glutamylcysteine-producing ability and is auxotrophic for pantothenic acid is proliferated in a medium containing a sufficient amount of pantothenic acid, and then it is cultured in a medium containing a limited amount of pantothenic acid to increase the γ-glutamylcysteine content in its cells, whereby the yeast in which γ-glutamylcysteine is accumulated is obtained.

## Claims

### Claim 1

An isolated yeast which has a γ-glutamylcysteine-producing ability and is auxotrophic for pantothenic acid, wherein γ-glutamylcysteine content per dried cells of the yeast increases in course of time when the yeast is cultured in a medium containing a limited amount of pantothenic acid,
wherein said yeast comprises a mutated endogenous chromosomal polynucleotide sequence encoding glutathione synthetase wherein said mutation reduces or eliminates gluthathione synthetase activity, and
wherein said yeast further comprises a mutated MET30 gene wherein the mutation replaces serine at position 569 of a protein encoded by the MET30 gene with phenylalanine and wherein expression of said mutated MET30 gene derepresses expression of the endogenous MET25 gene,
wherein said polynucleotide encoding said glutathione synthetase hybridizes with the polynucleotide of SEQ ID NO: 1 under stringent conditions,
wherein said MET30 gene hybridizes with the polynucleotide of SEQ ID NO: 3 under stringent conditions, and
wherein said stringent conditions comprise washing at 60° in 0.1×SSC and 0.1% SDS.

### Claim 2 (depends on 1)

The yeast according to claim 1, which belongs to the genus Saccharomyces.

### Claim 3 (depends on 1)

A method for producing a yeast in which γ-glutamylcysteine is accumulated, comprising the step to proliferate the yeast according to claim 1 by culturing it in a medium containing a sufficient amount of pantothenic acid, and the step to increase γ-glutamylcysteine content in the yeast cells by culturing it in a medium containing a limited amount of pantothenic acid.

### Claim 4 (depends on 1)

A food or beverage comprising a culture obtained by culturing the yeast according to claim 1 under a suitable condition.

### Claim 5 (depends on 1)

A method for producing a food or beverage containing γ-glutamylcysteine or cysteine, comprising culturing the yeast according to claim 1 under a suitable condition, mixing the obtained culture or fractionated product thereof, or the culture or fractionated product thereof subjected to a heat-treatment with a food or beverage raw material, and processing the mixture into the food or beverage.

### Claim 6 (depends on 4)

The food or beverage according to claim 4, which is selected from the group consisting of an alcoholic beverage, a bread food, and a fermented food flavoring material.

### Claim 7 (depends on 4)

The food or beverage according to claim 4, wherein said culture is heat treated to produce cysteine.

### Claim 8

A yeast in which expression of a MET25 gene is derepressed by harboring a mutant MET30 gene having a mutation which replaces serine at position 569 of a protein encoded by the MET30 gene with phenylalanine, wherein said MET30 gene hybridizes with the polynucleotide of SEQ ID NO: 3 under stringent conditions, and wherein said stringent conditions comprise washing at 60° C. in 0.1×SSC and 0.1% SDS.